This invention relates to the use of an enzyme for the oxidation or reduction of pyridine nucleotide cofactors during enzymic reactions in vivo or in vitro, for example in enzymic or whole-cell biotransformations or enzymic analytical techniques.
Biotransformation procedures using natural or genetically-modified microorganisms or isolated enzymes provide methods for the synthesis of many useful products. Biotransformations have several advantages over chemical synthetic methods, in particular regiospecificity and stereospecificity of the enzyme-catalysed reactions, use of mild reaction conditions, and absence of requirement for toxic solvents.
Oxidoreductase enzymes often require redox-active cofactors for activity. Among the most common such cofactors are the pyridine nucleotide cofactors nicotinamide adenine dinucleotide (NAD: oxidized form NAD+, reduced form NADH) and nicotinamide adenine dinucleotide phosphate (NADP: oxidized form NADP+, reduced form NADPH). These cofactors are expensive and, except in the cases of extremely valuable products, cannot feasibly be supplied in stoichiometric quantities. This is one factor limiting the use of many oxidoreductase enzymes for biotransformation reactions.
The requirement for cofactors in a biotransformation process can be reduced by the provision of a means of regenerating the desired form of the cofactor. This means that the cofactor need be supplied only in catalytic quantities. For example, if the reaction of interest requires NAD+, which is reduced in the reaction to NADH, the NADH can be re-oxidized by NAD+ by another enzyme system, such as NAD+-dependent formic dehydrogenase in the presence of formate. This is referred to as cofactor cycling. Formic dehydrogenase is particularly suitable for this purpose, since the reaction it catalyses is essentially irreversible.
A further complication is that the majority of NAD-requiring enzymes are not able to use NADP as a cofactor, and vice versa. For example, formic dehydrogenase could not be used to regenerate NADPH from NADP+.
A special case is where a biotransformation process requires two oxidoreductase enzymes which require different cofactors. For example, a recently proposed biotransformation process for the conversion of morphine to the powerful painkiller hydromorphone requires the sequential action of NADP+-dependent morphine dehydrogenase and NADH-dependent morphinone reductase (French et of (1995) Bio/Technology 13:674-676). In the first reaction, morphine is converted to morphinone with reduction of NADP+ to NADPH, and in the second reaction morphinone is converted to hydromorphone with oxidation of NADH to NAD+. Therefore, both NADP+ and NADH must be supplied. A further complication is that, in the presence of NADPH generated in the first reaction, morphine dehydrogenase reduces the product hydromorphone to an undesirable product, dihydromorphine, with re-oxidation of NADPH to NADP+. These reactions are shown in the accompanying FIG. 1A.
Pyridine nucleotide-dependent enzymes can also be used in certain enzymic assay procedures, with the quantity of the analyte being determined by the degree of oxidation or reduction of the cofactor. Oxidation and reduction of NAD and NADP can be measured by several methods; for example, spectrophotometry and fluorimetry. However, exceptionally sensitive methods for detecting oxidation or reduction may only be available for either NAD or NADP, but not both. For example, the oxidation of NADH to NAD+ can be detected with extreme sensitivity by using the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerokinase (PGK) to phosphorylate adenosine diphosphate (ADP) to adenosine triphosphate (ATP) in a reaction dependent on the presence of NAD+, and then detecting the resulting ATP by the ATP-dependent light-emitting reaction of firefly luciferase. This method cannot be used to detect oxidation of NADPH to NADP+, since the commercially available GAPDH is specific for NAD+.
Several of the problems mentioned above can be overcome by the use of an enzyme which transfers reducing equivalents between NAD and NADP; for example, reducing NAD+ to NADH while oxidizing NADPH to NADP+. Such an enzyme is known as a pyridine nucleotide transhydrogenase (PNTH). Several types of enzyme exhibit this activity (Rydstrxc3x6m et al (1987) in xe2x80x98Pyridine nucleotide coenzymes: chemical, biochemical and medical aspectsxe2x80x99, part B, eds. Dolphin et al, John Wiley and Sons, NY, p.433-460) The best known is the membrane-bound, proton-pumping, PNTH found in the membranes of mitochondria and certain bacteria such as Escherichia coli. This enzyme, being membrane-bound, is generally unsuitable for biotransformation and analytical purposes. Soluble, non-energy-linked PNTH has been reported to occur in certain bacteria such as Pseudomonas fluorescens, Pseudomonas aeruginosa and Azotobacter vinelandii. This enzyme has been characterized in some detail, but its utility is limited.
The gene (designated sth) encoding the soluble transhydrogenase of Pseudomonas fluorescens NCIMB 9815 has been cloned and sequenced, and the enzyme has been overexpressed in Escherichia coli. This enables the preparation of large amounts of enzyme relatively easily. The enzyme has been purified and characterized. This enzyme is defined by the reaction it catalyses, namely, transfer of reducing equivalents between NAD and NADP or analogues of these cofactors; the nucleotide sequence of the structural gene, sth, encoding the enzyme, and the deduced amino acid sequence of the enzyme derived therefrom; structural properties of the enzyme, including a subunit Mr of approximately 50,000; and the capacity to form large polymers of Mr exceeding 1,000,000.
According to a first aspect of this invention, the enzyme is used to act upon pyridine nucleotide cofactors so as to enhance a biotransformation process, for example, to alter the oxidation state of NAD or NADP or analogues of these cofactors. This may be so as to allow the action of another enzyme upon these cofactors. Alternatively, an altered form of the enzyme, prepared by random or site-directed mutagenesis of the structural gene, might be used. Such an altered enzyme may show altered levels of activity, altered regulation, or altered subunit structure.
The gene sth constitutes a second aspect of this invention. The gene may be used for the production of the enzyme or an altered form of the enzyme using a genetically modified organism. For example, a genetically modified organism carrying the sth gene as all or part of a heterologous construct may be grown in such a way as to encourage production of the enzyme, which may then be recovered from the culture medium or from cell extracts. The methods for accomplishing this are well known in the art.
A third aspect of this invention is the genetically modified organism which expresses the enzyme. Such an organism may be used in a whole cell biotransformation process which may be enhanced by the presence in the cells of the active enzyme. Techniques for generating such recombinant organisms are well known in the art.
According to a fourth aspect of the invention, the enzyme is used in enzyme-based analytical assays so as to enhance these assays. For example, the enzyme may be used to, in effect, convert a signal measured as oxidation of NADPH to NADP+ to a signal that can be measured based on oxidation of NADH to NAD+. The altered signal may thereafter be detected by a more sensitive technique which was not formerly applicable.